The present invention relates to transgenic animals, compositions and methods relating to the characterization of gene function.
Many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers such as cAMP. The membrane protein gene superfamily of G-protein coupled receptors (GPCRs) include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors. GPCRs have been characterized as having seven putative transmembrane domains (designated TM1, TM2, TM3, TM4, TM5, TM6, and TM7), which are believed to represent transmembrane xcex1-helices connected by extracellular or cytoplasmic loops. Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops that form disulfide bonds that are believed to stabilize functional protein structure. G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters. Different G-protein xcex1-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell.
One important subfamily of the GPCRs is the neuropeptide Y receptor (NPYR) GPCRs. Neuropeptides are small peptides originating from large precursor proteins synthesized by peptidergic neurons and endocrine/paracrine cells, and hold promise for treatment of neurological, psychiatric, and endocrine disorders. Often, the precursors contain multiple biologically active peptides. There is great diversity of neuropeptides in the brain caused by alternative splicing of primary gene transcripts and differential precursor processing. The neuropeptide receptors serve to discriminate between ligands and to activate the appropriate signals.
Neuropeptide Y (NPY), the most abundant neuropeptide to be identified in mammalian brain, is a 36 amino acid peptide which belongs to a family of neuroendocrine peptides consisting of NPY, peptide YY (PYY) and pancreatic polypeptide (PP). It is widely distributed throughout the central and peripheral nervous systems of mammals (Gen. Pharmacol. 24, 785 (1993); Cardiovasc. Res. 27, 893 (1993)). In the brain, NPY is particularly abundant in the hypothalamus, the limbic system and the cortex (Neurosci. 15, 1149 (1985)). In the periphery, it is localized in sympathetic nerve fibers that surround blood vessels and other smooth muscle tissues. NPY exerts a remarkably wide variety of physiological effects of potential therapeutic importance. It induces vasoconstriction when administered alone and acts synergistically when administered with other vasoconstrictors such as KC1, ATP, angiotensin II and histamine (Ann. NY Acad. Sci. 611, 7 (1990); Ann. NYAcad. Sci. 611, 166 (1990); Am. J. Physiol. 258, R736 (1990); and Gen. Pharmacol. 20, 363 (1989)). When acting upon coronary arteries, the vasoconstrictive action of NPY can cause angina pectoris (Lancet 1(8541), 1057 (1987)). In addition, NPY has been found to restore the response to vasoconstrictors after desensitization which follows multiple exposures to vasoactive substances or after endotoxic shock (Am. J. Physiol. 265, H1416 (1993)). NPY also exerts a mitogenic effect on aortic and venous smooth muscle tissue, and may contribute to cardiovascular hypertrophy in hypertension. Recent data suggests that it may promote angiogenesis as efficiently as basic fibroblast growth factor (Peptides 14, 263 (1993)). A third physiological action of NPY is in the hypothalamic regulation of body temperature, energy balance and metabolism. There is a large body of evidence indicating that NPY induces food intake in animals when injected in the hypothalamic area (Endocrinol. 115, 427 (1984); Life Sci. 35, 2635 (1984); Peptides 5, 1025 (1984)). Recent reports suggest that it is the major mediator of the action of OB/leptin, a protein which acts centrally to reduce food consumption (Nature 377, 530 (1995)), and that it has a direct anti-lipolytic effect on adipocytes (Am. J. Physiol. 265, E74 (1993)). Other results suggest that NPY may be important in the treatment of some forms of type II diabetes (Diabetes 40, 660 (1991); Regul. Peptides 34, 225 (1991)). Intracerebroventricular injection of the peptide enhances insulin secretion from pancreatic islets via autonomic control, whereas, in the periphery, NPY has a direct inhibitory effect on pancreatic insulin release. Still another physiological effect reported for NPY is in the regulation of gonadotropin secretion. There are indications that it may play a role in follicular maturation and ovulation (Biochem. Biophys. Res. Comm. 200, 1111 (1994); Ann. NY Acad. Sci. 611, 273 (1990); Neuropep. 30, 293 (1996)). It has been found that NPY levels are elevated in rats with decreased sexual function and that ventral administration of the peptide reduces sexual performance. In addition, several lines of evidence indicate that sex steroids exert a feedback regulation on NPY levels (Endocrinol. 130, 3331 (1992); Endocrinol. 132, 139 (1993)). Still other effects of NPY include: improved memory retention as observed in mice (J. Pharmacol. Exp. Ther. 268, 1010 (1994)); analgesia in animal models of pain (Brain Res. 724, 25 (1996)); inhibition of the excitatory amino acid glutamate, suggesting a possible role in epilepsy (Br. J. Pharmacol. 113, 737 (1994)); and modulation of nasal vasodilation, rhinorrhea and bronchial secretion, suggesting possible importance in treating allergic rhinitis and cystic fibrosis (Br. J. Pharmacol. 118, 2079 (1996); Am. J. Physiol. 266, L513 (1994)).
Reduced cortical concentrations of NPY have been observed in animal models of depression, and antidepressants have been found to increase NPY production (Clin. Neurophannacol. 9(4), 572 (1986)). NPY has been reported to produce an anxiolytic effect in animal models of anxiety (Psychopharmacol. 98, 524 (1989)). In addition, concentrations of NPY are reduced in the CSF of patients with major depression or severe anxiety and in the brain tissue of some suicide victims (Annu. Rev. Pharmacol. Toxicol. 32, 309 (1993)).
At least six distinct subtypes of NPY receptors have been described. The receptor subtypes, named Y1, Y2, etc., were initially classified based upon their selectivity for NPY, PYY and PP, as well as for their binding to NPY analogues and C-terminal fragments.
Recently, the complete cds of a murine pancreatic neuropeptide Y receptor gene, NPY6-R (aka pancreatic polypeptide receptor 2, or PP2), has been reported to be cloned and expressed from mouse genomic DNA (J. Biol. Chem. 271(28):16435-8 (1996)). In situ hybridization of mouse brain sections revealed expression of this receptor within discrete regions of the hypothalamus including the suprachiasmatic nucleus, anterior hypothalamus, bed nucleus stria terminalis, and the ventromedial nucleus with no localization apparent elsewhere in the brain. The NPY6-R sequence (GenBank GI or NID number: 1378003; Accession number: U58367) comprises 2281 bp; the coding region is believed to comprise bases 823-1938.
More recently, a transgenic mouse with a disrupted NPY-Y1 gene has been reported (U.S. Pat. No. 5,817,912); this xe2x80x9cknockoutxe2x80x9d mouse was studied with respect to eating behavior and weight changes.
Given the importance of GPCRs, particularly the neuropeptide receptors, a clear need exists for identification and characterization of GPCRs which can play a role in preventing, ameliorating or correcting dysfunctions or diseases.
The present invention generally relates to transgenic animals, as well as to compositions and methods relating to the characterization of gene function.
The present invention provides transgenic cells comprising a disruption in a NPY6 receptor (NPY6-R) gene. The transgenic cells of the present invention are comprised of any cells capable of undergoing homologous recombination. Preferably, the cells of the present invention are stem cells and more preferably, embryonic stem (ES) cells, and most preferably, murine ES cells. According to one embodiment, the transgenic cells are produced by introducing a targeting construct into a stem cell to produce a homologous recombinant, resulting in a mutation of the NPY6 receptor gene. In another embodiment, the transgenic cells are derived from the transgenic animals described below. The cells derived from the transgenic animals includes cells that are isolated or present in a tissue or organ, and any cell lines or any progeny thereof.
The present invention also provides a targeting construct and methods of producing the targeting construct that when introduced into stem cells produces a homologous recombinant. In one embodiment, the targeting construct of the present invention comprises first and second polynucleotide sequences that are homologous to the NPY6 receptor gene. The targeting construct also comprises a polynucleotide sequence that encodes a selectable marker that is preferably positioned between the two different homologous polynucleotide sequences in the construct. The targeting construct may also comprise other regulatory elements that may enhance homologous recombination.
The present invention further provides non-human transgenic animals and methods of producing such non-human transgenic animals comprising a disruption in a NPY6 receptor gene. The transgenic animals of the present invention include transgenic animals that are heterozygous and homozygous for a mutation in the NPY6 receptor gene. In one aspect, the transgenic animals of the present invention are defective in the function of the NPY6 receptor gene. In another aspect, the transgenic animals of the present invention comprise a phenotype associated with having a mutation in a NPY6 receptor gene.
The present invention also provides methods of identifying agents capable of affecting a phenotype of a transgenic animal. For example, a putative agent is administered to the transgenic animal and a response of the transgenic animal to the putative agent is measured and compared to the response of a xe2x80x9cnormalxe2x80x9d or wild type mouse, or alternatively compared to a transgenic animal control (without agent administration). The invention further provides agents identified according to such methods. The present invention also provides methods of identifying agents useful as therapeutic agents for treating conditions associated with a disruption of the NPY6 receptor gene.
The present invention further provides a method of identifying agents having an effect on NPY6 receptor expression or function. The method includes administering an effective amount of the agent to a transgenic animal, preferably a mouse. The method includes measuring a response of the transgenic animal, for example, to the agent, and comparing the response of the transgenic animal to a control animal, which may be, for example, a wild-type animal or alternatively, a transgenic animal control. Compounds that may have an effect on NPY6 receptor expression or function may also be screened against cells in cell-based assays, for example, to identify such compounds.
The invention also provides cell lines comprising nucleic acid sequences of a NPY6 receptor gene. Such cell lines may be capable of expressing such sequences by virtue of operable linkage to a promoter functional in the cell line. Preferably, expression of the NPY6 receptor gene sequence is under the control of an inducible promoter. Also provided are methods of identifying agents that interact with the NPY6 receptor gene, comprising the steps of contacting the NPY6 receptor gene with an agent and detecting an agent/NPY6 receptor gene complex. Such complexes can be detected by, for example, measuring expression of an operably linked detectable marker.
The invention further provides methods of treating diseases or conditions associated with a disruption in a NPY6 receptor gene, and more particularly, to a disruption in the expression or function of the NPY6 receptor gene. In a preferred embodiment, methods of the present invention involve treating diseases or conditions associated with a disruption in the NPY6 receptor gene""s expression or function, including administering to a subject in need, a therapeutic agent that effects NPY6 receptor expression or function. In accordance with this embodiment, the method comprises administration of a therapeutically effective amount of a natural, synthetic, semi-synthetic, or recombinant NPY6 receptor gene, NPY6 receptor gene products or fragments thereof as well as natural, synthetic, semi-synthetic or recombinant analogs.
The present invention further provides methods of treating diseases or conditions associated with disrupted targeted gene expression or function, wherein the methods comprise detecting and replacing through gene therapy mutated NPY6 receptor genes.
Definitions
The term xe2x80x9cgenexe2x80x9d refers to (a) a gene containing at least one of the DNA sequences disclosed herein; (b) any DNA sequence that encodes the amino acid sequence encoded by the DNA sequences disclosed herein and/or; (c) any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein. Preferably, the term includes coding as well as noncoding regions, and preferably includes all sequences necessary for normal gene expression including promoters, enhancers and other regulatory sequences.
The terms xe2x80x9cpolynucleotidexe2x80x9d and xe2x80x9cnucleic acid moleculexe2x80x9d are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term xe2x80x9cpolynucleotidexe2x80x9d includes single-, double-stranded and triple helical molecules. xe2x80x9cOligonucleotidexe2x80x9d refers to polynucleotides of between 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. A xe2x80x9cprimerxe2x80x9d refers to an oligonucleotide, usually single-stranded, that provides a 3xe2x80x2-hydroxyl end for the initiation of enzyme-mediated nucleic acid synthesis. The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.
A xe2x80x9cfragmentxe2x80x9d of a polynucleotide is a polynucleotide comprised of at least 9 contiguous nucleotides, preferably at least 15 contiguous nucleotides and more preferably at least 45 nucleotides, of coding or non-coding sequences.
The term xe2x80x9cgene targetingxe2x80x9d refers to a type of homologous recombination that occurs when a fragment of genomic DNA is introduced into a mammalian cell and that fragment locates and recombines with endogenous homologous sequences.
The term xe2x80x9chomologous recombinationxe2x80x9d refers to the exchange of DNA fragments between two DNA molecules or chromatids at the site of homologous nucleotide sequences.
The term xe2x80x9chomologousxe2x80x9d as used herein denotes a characteristic of a DNA sequence having at least about 70 percent sequence identity as compared to a reference sequence, typically at least about 85 percent sequence identity, preferably at least about 95 percent sequence identity, and more preferably about 98 percent sequence identity, and most preferably about 100 percent sequence identity as compared to a reference sequence. Homology can be determined using a xe2x80x9cBLASTNxe2x80x9d algorithm. It is understood that homologous sequences can accommodate insertions, deletions and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide residues do not precisely correspond or align. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome.
The term xe2x80x9ctarget genexe2x80x9d (alternatively referred to as xe2x80x9ctarget gene sequencexe2x80x9d or xe2x80x9ctarget DNA sequencexe2x80x9d or xe2x80x9ctarget sequencexe2x80x9d) refers to any nucleic acid molecule or polynucleotide of any gene to be modified by homologous recombination. The target sequence includes an intact gene, an exon or intron, a regulatory sequence or any region between genes. The target gene comprises a portion of a particular gene or genetic locus in the individual""s genomic DNA. As provided herein, the target gene of the present invention is a NPY6 receptor gene. A xe2x80x9cNPY6 receptor genexe2x80x9d or xe2x80x9cNPY6-R genexe2x80x9d as used herein refers to a sequence comprising SEQ ID NO: 1, i.e., comprising the sequence identified in GenBank as GI or NID number: 1378003; Accession number: U58367.
xe2x80x9cDisruptionxe2x80x9d of a NPY6 receptor gene occurs when a fragment of genomic DNA locates and recombines with an endogenous homologous sequence. These sequence disruptions or modifications may include insertions, missense, frameshift, deletion, or substitutions, or replacements of DNA sequence, or any combination thereof. Insertions include the insertion of entire genes, which may be of animal, plant, fungal, insect, prokaryotic, or viral origin. Disruption, for example, can alter or replace a promoter, enhancer, or splice site of a NPY6 receptor gene, and can alter the normal gene product by inhibiting its production partially or completely or by enhancing the normal gene product""s activity.
The term, xe2x80x9ctransgenic cellxe2x80x9d, refers to a cell containing within its genome a NPY6 receptor gene that has been disrupted, modified, altered, or replaced completely or partially by the method of gene targeting.
The term xe2x80x9ctransgenic animalxe2x80x9d refers to an animal that contains within its genome a specific gene that has been disrupted by the method of gene targeting. The transgenic animal includes both the heterozygote animal (i.e., one defective allele and one wild-type allele) and the homozygous animal (i.e., two defective alleles).
As used herein, the terms xe2x80x9cselectable markerxe2x80x9d or xe2x80x9cpositive selection markerxe2x80x9d refers to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance (Neor) gene are resistant to the compound G418. Cells that do not carry the Neor gene marker are killed by G418. Other positive selection markers will be known to those of skill in the art.
A xe2x80x9chost cellxe2x80x9d includes an individual cell or cell culture that can be or has been a recipient for vector(s) or for incorporation of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent due to natural, accidental, or deliberate mutation. A host cell includes cells transfected with the constructs of the present invention.
The term xe2x80x9cmodulatesxe2x80x9d as used herein refers to the inhibition, reduction, increase or enhancement of a NPY6 receptor function, expression, activity, or alternatively a phenotype associated with a disruption in a NPY6 receptor gene. In a preferred embodiment, the term xe2x80x9cmodulatesxe2x80x9d refers to agents that act as agonists or antagonists of the receptor.
The term xe2x80x9camelioratesxe2x80x9d refers to a decreasing, reducing or eliminating of a condition, disease, disorder, or phenotype, including an abnormality or symptom associated with a disruption in a NPY6 receptor gene.
The term xe2x80x9cabnormalityxe2x80x9d refers to any disease, disorder, condition, or phenotype in which a disruption of a NPY6 receptor gene is implicated, including pathological conditions.